Increasingly, biological fragment separations demand cost-effective high-throughput, high-performance technologies. Sample fragment separations using slab gel technology has been supplanted by capillary array electrophoresis (CAE). The throughput of a CAE system is directly proportional to the number of separation capillaries in the instrument. However, as the number of capillaries increases, it becomes more challenging to control sample injection and to detect signals from all of the capillaries.
Another technology for high-throughput DNA analysis is capillary array electrophoresis on microchips. Microchips are planar members typically formed from a glass, silica, or even polymeric material. Photolithographic techniques are typically used to microfabricate CAE channels on substrates. The microchip substrate defines at least one elongate capillary channel which extends between opposed cathode and anode ports. Sample and waste ports are located adjacent the cathode port and channel segments extend therefrom to the elongate micro-channel. D. Harrison et al., Anal. Chem. 64, 1926-1932; Z. Fan et al., Anal. Chem. 66, 177-184; and S. Jocobson et al., Anal. Chem. 66, 1107-1113. When a biological, fluid sample is deposited in the sample port, electrical potential may be applied to the four ports so as to direct a portion of the fluid sample first into the elongate micro-channel and then towards the opposed anode port. These design is often called ‘T’ or “twin T” injection scheme. The fluid sample, which separates into different-length segments, is analyzed as it passes a point in the channel at which is read by an interrogation device. Microchips have been used in separation of not only biological samples, but also chemicals as well. For example, microchips have been used to separate fluorescent dyes, fluorescently-labeled amino acids, DNA restriction fragments, PCR products, short oligonucleotides, short tandem repeats, and DNA sequencing fragments.
In order to increase throughput, multiple CAE channels have been microfabricated on microchips and used for DNA fragment size analysis. Channels on many substrate designs include right angle turns that work well for fragment sizing but which degrade performance in sequencing separations. Alternate designs, using a round substrate, include radially-extending channels terminating at a common, centrally-located anode. For example, Shi et al. in Anal. Chem. 1999, 71, 5354-5361, disclose a 96 channel radial CAE microchip design for use with a rotary confocal fluorescence detection system. The 96 channels are formed on a 10 centimeter diameter Borofloat substrate so as to extend from a common, centrally-located anode. Such a design makes effective use of the chip space in providing uniform-length channels while still allowing a detector to scan perpendicularly across all of the channels. One drawback to this design, however, is that the effective channel lengths are limited to less than one-half of the chip diameter, or here to 3.3 centimeters for a 10 centimeter diameter chip. The effective channel length refers to the distance a fluid would travel through a channel before reaching the point where it is interrogated by an analytical device. While channels of this length work well for separations of certain restriction fragments and genotyping samples, it is very challenging to achieve sequencing separations using such short channels. In order to increase the length of the channels, larger-diameter chips may obviously be used, however the fabrication costs of suitable larger chips can be cost-prohibitive.
One of the limitations of the current micro-channel plate (MCP) design for chemical/biological sample separation lies in the maximum density of channels on a plate. The area of the interface holes limits the channel density on the actual chips, therefore the throughput of separation and the detection. Such a limitation is intrinsic to the horizontal “T” or “double T” injection design.
In the case of short channels on chips, the injected sample plug properties are essential for a good separation. It has to be short enough to produce a good resolution and concentrated enough for a processable signal.
The use of the conventional pipette or syringe based fluid transfer method for sample loading to the MCP channel is another area needs improvement. The matrix in the sample well has to be removed before sample loading. Fluid control equipment is required to clean the tip of the pipette. In practice, this system proves to be complex, as well as costly.
There is therefore a need in the art for a cost-effective high-throughput, high-performance electrophoresis microchip which maximizes formation of uniform-length, elongate electrophoresis separation microchannels thereon. There is also a need in the art for an electrophoresis microchip which provides a compact array of microchannels so as to increase throughput. There is still another need of improved sample injections methods.